The purpose of this experiment is to extract DNA from the plant cells in a strawberry. Every living organism contains DNA. Deoxyribonucleic acid (DNA) is the genetic material of a living organism and it is in the chromosomes of each cell. Strawberries are octoploid and have eight copies of each chromosome per cell and make it easier to quantify an amount from a sample. The basic steps in DNA extraction involve breaking open cells to release the DNA followed by the collection of the DNA by alcohol precipitation. The isolation and purification of DNA is a key step for most protocols in molecular biology.
The cells are chemically treated using a lysis buffer containing detergent and sodium chloride. This detergent breaks open the cell walls by destroying phospholipid bilayers of cell membranes, nuclear membranes, and organelles releasing the DNA and other cell contents. The salt binds with the negatively charged phosphate groups in the DNA which will cause precipitation of the alcohol in high concentration alcohol.
Start by weighing 5-8 grams of strawberry noting the exact mass. Place the weighed amount of strawberry inside a plastic bag and mechanically break down the cell walls and structure by smashing the berry into a mush. Now, add 8ml of lysis buffer to a small beaker. Squeeze the mush out of the bag and mix thoroughly to help with the chemical extraction of the DNA by lysing the cell membranes. Next, separate the liquid and solid particles from the solution by pouring the mixture through a funnel with cheesecloth as a filter and collecting the filtrate in a clean beaker. The solid mass with cheesecloth can be discarded. Measure the filtrate and add carefully an equal amount of ice-cold 70% propanol to create a mixture. Special care should be taken not to mix the filtrate with ice-cold propanol. Allow the mixture to continue separating and settling by letting it cool in a fridge for 10 minutes. Remove the mixture from the fridge carefully not to mix the contents and extract the DNA. The tube contents should have two distinct layers with a bundle strand of DNA in-between them located in the interface zone. Place the spooled DNA pellet into a microtube containing ice-cold 70% propanol and centrifuge for 20 seconds. Pour of the isopropanol without disturbing the pellet. Now add 500 microliters of ice-cold 70% propanol to the microtubule and vortex to mix. Pour off the isopropanol again and add 100 microliters of distilled water to the tube sand vortex to mix. Set the sample in the fridge and allow the DNA to dissolve for one week. After one week, transfer 50 microliters of your DNA sample to a small tube and add 450 microliters of distilled water and mix well. Transfer the diluted DNA solution into a quartz cuvette. By using a spectrometer set at 260 nanometers, the optical density (OD)can be read to calculate the concentration of DNA calculate the total DNA yield of your preparation and the yield per gram of sample.
Results and discussion
In this experiment, we successfully extracted DNA from a 8.619g sample of strawberry, and could observe visible strands of DNA. The extraction of DNA was facilitated by the presence of eight copies of each chromosome in the strawberry. In contrast, humans have a diploid genome, which includes only two copies of each chromosome. The strawberry was degraded by crushing it and mixing it with 8mL of lysis buffer which cause the cell walls, cell membranes, and nuclear membranes to separate. The mashing physically disrupted the cell walls, and the buffer dissolved the membrane phospholipid bilayers, and the salt provided optimum osmolarity. The large solids were then separated by filtration, and the resulting solution was collected in a test tube. At that time, the DNA was dissolved in water, which is a polar solvent; and sodium ions bonded with the negatively charged DNA via an ionic bond. After the addition of ethanol, DNA precipitated into the nonpolar layer, which made the long DNA strands easily visible to the naked eye. The DNA was then stored in the fridge for one week. The next week in lab 50uL of DNA had to be added into a small tube as well as 450uL of water had to be mixed in the tube with the DNA. Next the tube had to be placed into a spectrophotometer and read the optical density at 260nm. After that is complete the DNA concentration and the DNA yield could be calculated. The concentration was 127.628ug/mL and the yield was 12.7628ug. which means there was 1.48ug DNA per gram of strawberry.
The purpose of this experiment was to extracted DNA from a strawberry as well as calculate the strawberry DNA concentration. When the DNA was extracted what was found was much greater than expected. There was more than likely errors that happened throughout the experiment and to be able to insure the results the experiment would need to be repeated a couple of times. When doing this experiment in the future students should be more careful while doing the extraction and make sure not to containment it. Also when the spectrometer is needed make sure it is properly calibrated so the results are correct. When the experiment is completed if it is done correctly the yield of DNA would be greater.
...(download the rest of the essay above)